Dental caries, although largely preventable, is the most common chronic disease for humans from ages 6 - 19 years. Untreated, caries will result in pulpal pathologies involving severe dental pain, and eventually tooth loss because dental enamel cannot regenerate. Enamel is an almost fully mineralized tissue in the vertebrate body composed of a substituted hydroxyapatite (Hap) of primarily calcium (Ca2+) and inorganic phosphate (Pi), yet researchers have not been able to define the molecular pathways involved in the transportation of Pi to the forming enamel or dentin extracellular matrix. Recent papers suggest that Ca2+ and bicarbonate (HCO3-) ions (the latter being a buffering agent that is important in Hap formation) are transported from the circulating blood, and from cells of the enamel organ papillary layer, into polarized ameloblasts through ion channels located at their basolateral membrane. They are then exported through a different series of ion channels located at the apical membrane, to be delivered to the enamel matrix. Mutations in many of the ion channels associated with Ca2+ and HCO3- transport result in enamel pathology. While initial studies are starting to define Ca2+ and HCO3- transcellular transport in amelogenesis, there is scant information related to coupled Pi transport. We have recently performed a whole-genome array analysis for secretory- and maturation-stage enamel organ cells, and this data identified that sodium-dependent Pi transporters feature prominently in amelogenesis. Our array data indicate that SLC20 and SLC34 gene family members are expressed at high levels throughout amelogenesis, and in particular the expression of one member, Slc34a2, increases greater than 50-fold as enamel cells transition from secretory- to maturation-stage. The hypothesis of this application is that transcellular transport of inorganic phosphate is critical o enamel formation, and Slc20a1, Slc20a2 and Slc34a2 are responsible for much of the Pi uptake in ameloblast cells throughout amelogenesis. To test this hypothesis we propose to: 1) define the mRNA and protein expression levels of SLC20 and SLC34 gene family members in secretory-stage and maturation-stage enamel organ cells; 2) define spatiotemporal localization profiles in enamel organ cells throughout amelogenesis for the most highly expressed SLC20 and SLC34 gene family members; and 3) investigate Pi uptake kinetics in ameloblast-like cell lines. Understanding how Pi is transported from the circulation to the enamel extracellular matrix is fundamental to dental health. If successful, investigators will better understand the molecular process of transcellular Pi transport events in ameloblasts, and future studies may identify molecular-based approaches to prevent, delay or repair damage to dental hard tissues from caries.